Enhancing Flucytosine Anticandidal Activity Using PEGylated Squalene Nanocarrier

Abstract

There is an emerging necessity for improved therapies against Candida-related infections, with significant implications for global healthcare. Current antifungal agents, limited in number, target specific pathways, but resistance remains a concern. Flucytosine (5FC) exhibits antifungal activity, particularly against Candida. However, monotherapy efficacy is limited, necessitating combination treatments. Herein, we report PEGylated squalene-based nanocarriers for 5FC loading, aiming to enhance its monotherapy efficacy against Candida strains. The loading of 5FC within micelles was achieved using the ultrasound-assisted solvent evaporation method. The 5FC-loaded micelles, together with non-loaded micelles, were thoroughly characterized and analyzed. STEM and DLS analysis confirmed the core-shell morphology with nanometric dimensions along with improved colloidal stability. The quantification of drug loading efficiency and drug loading capacity was calculated using the UV-Vis technique. The in vitro drug-release studies in simulated physiological conditions showed sustained release within 48 hours. Moreover, the release kinetics calculated using mathematical models showed a Fickian diffusion drug release mechanism in simulated physiological conditions with a slower diffusion rate. The in vitro antifungal activity was tested on Candida albicans, Candida glabrata, and Candida parapsilosis. The results showed improved antifungal activity for the nanotherapeutic and unchanged in vitro toxicity toward normal cells, suggesting promising advancements in 5FC therapy.

Keywords: Antifungal activity; Candida infections; Drug delivery; Flucytosine; Micelles.

Scheme 1

The experimental procedure for obtaining SQ‐PEG₁₅₀₀‐NH‐Boc amphiphilic copolymer.

Scheme 2

Schematic representation of the experimental procedure for obtaining 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles by ultrasound assisted solvent evaporation method.

Figure 1

DLS analysis of non‐loaded and 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles at 0.75 mg/mL in PBS with pH value of 7.4 at ambient temperature of 23 °C. a) Particle hydrodynamic diameter (Hd) distributions; b) Zeta Potentials. All the measurements were done in triplicates and the plots are representing the average values without error bars.

Figure 2

STEM morphological analysis of the studied samples. a) STEM image of non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles at 500 nm scale; b) size distribution plot of non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles (n=120 micelles); c) STEM image of 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles at 1 μm scale; d) size distribution plot of 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles (n=120 micelles).

Figure 3

UV‐Vis analysis of the studied samples: a) overlapping the absorbance spectra of non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles, free 5FC and 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles recorded in PBS solutions with pH value of 7.4; b) UV‐Vis spectra of 5FC in PBS with pH 7.4 at concentrations between 0.625 and 30.00 μg/mL; c) linear fitting of the absorbance recorded at 276 nm as function of 5FC concentration (μg/mL) in PBS 7.4.

Figure 4

In vitro cumulative release of 5FC from free 5FC solution and 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles. a) Cumulative release of 5FC (%) for 96 hours in simulated physiological conditions (PBS with pH of 7.4 and 37 °C); b) Cumulative release of 5FC (%) for 48 hours in simulated physiological conditions (PBS with pH of 7.4 and 37 °C); c) Cumulative release of 5FC (%) for 96 hours in mQ‐H₂O at 37 °C; d) Cumulative release of 5FC (%) for 48 hours in mQ‐H₂O at 37 °C. The experiment was carried out for 96 hours and the results are expressed as means±SD (n=3). *p <0.05, **p <0.01, ***p <0.001 and #p >0.05 (ns) by Student's t‐test.

Figure 5

Linear fitting of the mathematical models applied for the release of loaded 5FC in SQ‐PEG₁₅₀₀‐NH‐Boc micelles and free 5FC in simulated physiological conditions and free release conditions: a) Zero‐order model; b) First‐order model; c) Higuchi model, d) Hixson‐Crowell model; and e) Korsmeyer‐Peppas model.

Figure 6

The microbial growth inhibition: a) C. albicans, b) C. glabrata, c) C. parapsilosis expressed as percent of viable yeast colonies vs. incubation time; d) yeast colonies of the three strains after 40 minutes of incubation with free 5FC and 5FC‐loaded micelles.

Figure 7

In vitro High Content Screening (HCS) analysis of the studied samples after 24 hours of exposure. a) Dead cells quantitative estimation after the treatment with the studied samples; b) live cells quantitative estimation after the treatment with the studied samples; c) cell proliferation and viability under the sample's treatment. Cell nuclei in blue, cytoplasm of living cells in green, dead cells nuclei or remnants of nuclei in red.